Tc and re labeler radioactive glycosylated octreotide derivatives

ABSTRACT

Improved sst-receptor binding peptidic ligands for diagnostic and therapeutic applications in nuclear medicine are provided. The improved ligands contain either natural or unnatural amino acids or peptidomimetic structures that are modified at either the N-terminal or the C-terminal end or at both termini, a carbohydrate unit and a chelator or prosthetic group to provide a complexation of a radioisotope binding or holding the radioisotope. The sst- or SSTR-receptor binding peptidic ligands may also contain one or more multifunctional linker units optionally coupling the peptide, and/or the sugar moiety and/or the chelator and/or the prosthetic group. Upon administering the ligand to a mammal through the blood system the ligand provides improved availability, clearance kinetics, sst-receptor targeting and internalization over the non-carbohydrated ligands.

The present invention relates to novel radioactive octreotidederivatives that are glycosylated and bind the somatostatin receptor.

These so-called SSTR-ligands are suitable for the in vivo targeting ofsomatostatin receptors and find broad application in nuclear medicin.

Essential parts of these molecules are sugar moieties conjugateddirectly or via linkers to the bioactive part of the molecule. Comparedto the corresponding non-carbohydrated derivatives, these derivatives,labeled with radioisotopes such as Rhenium and Technetium, either viadirect reductive methods or via tricarbonyl complexes, lead to potentsomatostatin receptor ligands with improved tumor/non-tumor accumulationratios, improved pharmacokinetics and improved internalization kinetics.

The somatostatin receptor binding peptidic ligand of this invention areprepared from natural or unnatural (prepared) ligands. These ligandsbear structural modifications at the N-terminal end or the C-terminalend or both termini. Said peptide ligands have affinity to sst-receptorsand are graphically represented by the structure:

wherein X indicates the C-terminus of the ligand. In the compositions ofthis invention wherein there may be multiple ligands where is permittedthe formation of di- and multimers by mixed C- and N-terminal couplingof the peptide chain. Thus, the scope of this invention cover homo-,dimer, homomultimers or different receptor binding structures,heterodimers, and heteromultimers. The di- and multimers can be formedby mixed C- and N-terminal coupling of the peptide chain.

The ligand composition of this invention optionally contains at leastone linker unit that can be multifunctional. Such linker unit allowscoupling together of the peptide, sugar moiety and chelator to the via acondensation-, acylation-, alkylation-, substitution- or additionreaction. Typical linker units comprise ligands taken from peptidic orother organic structures such as L- or D-amino acids such as lysine,ornithine serine, glutamic acid, aspartic acid, O-amine serine,mercaptopropionic acid, hydroyy carbonic acides, amino carbonic acids,halogen carbonic acids or polyamino acids.

The improved somatostatin receptor binding peptidic ligand of thisinvention comprises a carbohydrate, specifically a sugar such as amono-, di- and trisaccharide. Typical suitable sugars include glucose,galactose, maltose, mannose, maltrotriose and lactose coupled viacovalent linkage. That is, the sugar can be combined via the Maillardreaction and Amadori rearrangement, glycosidic linkage, alklation,allyation or coupled via complex formation after modification, that is,formation of carbohydrate isonitritles or carbohydrated phophates.

Another component of the improved somatostatin receptor binding peptidicligand of this invention is a chelator. Typical chelators are peptidicor non-peptidic structures suitable for mono- or multidentatecomlexation of radioisotopes of Tc and Re. The chelator useful incompositions of this invention comprise one or more (ligand andcoligand(s)) organic molecules containing any number of functionalgroups necessary to the complexation of the radiometals, depending onits oxidation state and complex geometry. Exemplary suitable chelatorsare, for example, histidine, picolylamine diacetic acid, hydroxynicotine amide (HYNIC), mercaptoacetyl-glycyl-glycyl-glycine (MAG3) andtetrapeptides.

To more clearly describe the improved somatostatin receptor bindingpeptidic ligand of this invention, reference is made to FIGS. 1A-1C. InFIG. 1A-1C there is shown, in graphical schematic form, the variousconfigurations into which the peptide ligands of this invention can beprepared. Other practical configurations may occur to those skilled inthis art in keeping with the teaching of this specification.

In FIG. 1A, FIG. 1B and FIG. 1C there is shown mono- di- and multimers(wherein n is an integer of 2 or more) containing a linker moiety, Lsugar moiety, S and a chelator, C capable of holding a radioisotope ofTc and Re.

In FIGS. 1A, 1B and 1C the termini of the peptide is designated byindicating the C-terminus by X, the opposite terminus being the Nterminus. Thus the pharmacophoric peptide is coupled with the C- orN-terminal end to the linker, chelator, etc. As indicated above, boththe peptide ligand and the linker (multifunctional) can comprise naturalor unnatural amino acids. Of course, the ligand (i.e. octreotide) willnot be employed as the linker in compositions of this invention.

In FIG. 1A there is shown various configurations of compoistions of thisinvention providing multimers of peptide ligands of two or more. Themultimers may comprise either identical receptor peptide bindingstructures (homodimers, homomultimers) or different receptor bindingstructures (heterodimers, heteromultimers).

In FIG. 1B, there is shown various configurations of mers some of whichcontain a linker unit (multifunctional) and four of which do not containa linker unit. Thus it is seen that a multifunctional linker unit isoptional in the compoisitions of this invention.

In FIG. 1C, there is shown dimers and multimers wherein multiple linkerunits are employed with varying orientation of the peptide and, ofcourse, multiple petptide ligands.

The dimers and multimers shown in FIGS. 1A and 1C can be formed viacovalent linkage of the linker or the peptide ligand to the chelator orformation of a complex between the linker and the chelator.

The invention is described in is illustrated in the non-limitingexamples that follow.

EXAMPLES Example 1

Rational Design of Peptide Radiopharmaceuticals: In Vitro StudiesDemonstrate a Synergistic Effect of Carbohydration and C-TerminalOxidation of Octreotide on Ligand Induced SST-₂ Internalization

Aims: Besides by its pharmacokinetics, the suitability of a receptorbased tracer for imaging and therapeutic purposes is mainly determinedby its pharmacodynamic profile. Aim of this study was to investigate theeffect of carbohydration of octreotide and octreotate on SSTR endo- andexocytosis (internalization and externalization) and reendocytosis(recirculation).

Methods: Internalization, externalization and recycling of[¹²⁵I]Tyr³-octreotide (TOC), [¹²⁵I]Tyr³-octreotate (TOCA) and theirGlucose-(Gluc) and Maltotriose-(Mtr) derivatives were studied usingconfluent monolayers of AR42J-cells (SSTR₂). The cells were incubatedwith the radioligand for up to 120 min (n=3). At each time point theactivity in the supernatant, the surface-bound and the internalizedactivity were determined and normalized to the values of TOC.Externalization and recycling was studied after an incubation time of120 min over 2 h. Specificity of ligand binding was studied in acompetition experiment with 10:M Sandostatin.

Results: After 2 h of incubation the amount of internalized ligand [%internalized TOC]was as follows: Mtr-TOC (35±4%)<Gluc-TOC (121%)<TOCA(154%)<Mtr-TOCA (549%)<Gluc-TOCA (637%). In the competition experimentinternalization of all compounds dropped to <0.1±0.02% (30 min) of theapplied radioactivity. In the externalization experiment that allowedrecycling of the ligands, TOCA and glycated TOCA's showed about ⅔ ofextracellularly located radioactivity compared to the experiment withoutrecycling, while about 80% were found for TOC, Gluc-TOC and Mtr-TOC.Carbohydration of TOC had no significant effect on the availability ofthe ligands on the cell surface, whereas surface concentration of TOCAand Mtr-TOCA is increased by a factor of 2.1 and 2.3 respectivelycompared to TOC. Gluc-TOCA shows a fivefold increase of the availabilityof the tracer on the cell surface compared to TOC. The internalizationrate (internalized/surface bound act.) of TOC is not significantlyaffected by glycosylation, whereas TOCA shows a 1.4 fold increase. ForGluc-TOCA and Mtr-TOCA we observed internalization rates of 186 and 171%compared to TOC.

Conclusion: Using AR42J-cells, carbohydration of TOCA led to asignificant increase in cell surface concentration and internalizationrate of the radioligands.

Example 2

Carbohydrated TC-99M-Octreotide Derivatives for Spect Synthesis,Radiolabelling and In Vivo Data

Over the past decade different approaches to ^(99m)Tc- and ¹⁸F-labelledoctreotide derivatives for SSTR-scintigraphy using SPECT and PET havebeen investigated. Decristoforo et al. showed that ^(99m)Tc-labelledHYNIC-Tyr³-octreotide with EDDA as a coligand has favourable biokineticsand high tumor uptake in mice. 2-[¹⁹F]fluoropropionyl-D-Phe¹-octreotide,the only ¹⁸F-labelled octreotide analog known so far, shows predominanthepatobiliary excretion, which is one drawback with respect to itsapplication for in vivo SSTR-imaging.

The inventors found that N-terminal glycosylation of radioiodinatedTyr³-octreotide (TOC) and its Thr⁸-derivative Tyr³-octreotate (TOCA)leads to a significant improvement of the biodistribution, i.e. to anincreased tumor accumulation. To investigate the general applicabilityof this principle, we synthesized and evaluated glycosylated octreotide-and octreotate-derivatives for ^(99m)Tc-labelling (FIG. 2).N-glycosylated derivatives of Lys⁰(N-His)-TOC (K⁰(H)TOC) were used asprecursors for ^(99m)Tc-labelling using the organometallic aquaion[^(99m)Tc(H₂O)₃(CO)₃]⁺-approach [1]. The peptide was synthesizedaccording to a standard Fmoc-SPPS protocol. Conjugation with glucose(Gluc) and maltotriose (Mtr) was performed via Amadori reaction.^(99m)Tc-labelling of the Lys⁵-deprotected peptides yielded[^(99m)Tc]Gluc-K⁰(H)TOC and [^(99m)Tc]Mtr-K⁰(H)TOC with radiochemicalyields >97% based on the aquaion.¹ Egli A. et al. J. Nucl. Med. 40: 1913-1917 (1999)

Biodistribution studies of the ^(99m)Tc-labelled derivatives (30 and 120min p.i.) were performed in AR42J-tumor bearing nude mice (n=3-4).Internalisation- and externalisation experiments were carried out usingthe same cell line.

An increase of internalisation was found for [^(99m)Tc]Gluc-K⁰(H)TOC bya factor of 2.3±0.8 and for [^(99m)Tc]Mtr-K⁰(H)TOC by a factor of3.6±0.4 compared to the reference [¹²⁵I]TOC. While [¹²⁵I]TOC is rapidlyexternalised from the cell upon incubation, [^(99m)Tc]Gluc-K⁰(H)TOCremains trapped within the cell up to 120 min.

The biodistribution of [^(99m)Tc]Gluc-K⁰(H)TOC and[^(99m)Tc]Mtr-K°(H)TOC 2 h p.i. are shown in Table 1. TABLE 1 Tissueaccumulation [% iD/g] Of [^(99m)Tc] Gluc-K⁰ (H) TOC And [^(99m)Tc]Mtr-K⁰ (H) TOC In AR42J tumor bearing Nude mice 2 h p.i. (n = 3-4)[^(99m)Tc] Gluc-K⁰ (H) TOC [^(99m)Tc] Mtr-K⁰ (H) TOC blood 1.49 ± 0.184.12 ± 0.87 liver 15.89 ± 2.70  14.34 ± 1.55  intestine 2.83 ± 0.28 2.02± 0.41 stomach 11.28 ± 2.74  6.22 ± 0.75 kidney 19.49 ± 2.22  17.97 ±2.76  muscle 0.22 ± 0.04 0.40 ± 0.10 adrenals 6.47 ± 1.46 4.94 ± 0.38pancreas 6.43 ± 2.09 3.32 ± 0.38 tumor 12.21 ± 0.96  14.02 ± 6.33 Both glycosylated compounds show high accumulation in sst₂ positivetissue 120 min p.i. We suppose that the comparatively high non-specificuptake in excretion organs as well as a delayed blood clearance are dueto the insufficient complexation of the ^(99m)Tc-core by the bidentatehistidine ligand. The remaining metal coordination site may be saturatedby thiol-containing native proteins in vivo, which can result in rapidtrapping of these complexes in the blood, the liver and other excretionorgans. The formation of saturated complexes of the[^(99m)Tc(H₂O)₃(CO)₃]⁺-aquaion with tridentate ligands such as N-Ac-Hisresult in a significant decrease of activity accumulation in non-tumortissue. It can be concluded, that the introduced tracer design based onthe combination of carbohydrate conjugation of octreotide(-tate) and oftridentate complexation of the [^(99m)Tc(H₂O)₃(CO)₃]⁺-aquaion is thebasis of a new series of very promising SSTR-tracers.

Example 3

Glycation of SST-Receptor-Agonists: Improvement of Dynamic LigandTrafficking of Radiolabelled Somatostatin Agonists

Carbohydration is now found to be a powerful tool to improve thepharmacokinetics of radiolabelled octreotide and ∀_(v∃3) analogs.Glycation of octreotide and octreotate significantly reduceshepatobiliary excretion and kidney uptake and enhances tumor uptake andtumor/tissue ratios. Tumor accumulation of a radiolabelled sst₂-agonistrelies upon the dynamic processes of receptor mediated internalisation,degradation and subsequent intracellular accumulation or recyling ofboth ligand and/or metabolite. Quantitative analysis of each step is ofcritical importance to understand how to control tracer accumulation insst₂ expressing tumor cells. Thus, the objective of this study was toexamine the effect of glycation on the internalization and recyclingkinetics of radiolabelled octreotide and octreotate analogues.

Tyr³-octreotide (TOC), Tyr³-octreotate (TOCA) and their respectiveglucose- (Gluc), maltose- (Malt) and maltotriose- (Mtr) derivatives weresynthesized via Fmoc-SPPS and subsequent carbohydrate conjugation.Radioiodination was performed using the iodogen method. Radiochemicalyields ranged from 50 to 84% after RP-HPLC-purification. Precursors for^(99m)Tc-labelling (Gluc-Lys⁰(N-His)TOC (Gluc-K(H)-TOC) andMtr-Lys⁰(N-His)TOC (Mtr-K(H)-TOC)) were prepared analogously to thepeptides mentioned above. Labelling with ^(99m)Tc was carried outaccording to the [[^(99m)Tc) (CO)₃(H₂O)₃]⁺ aquaion protocol previouslydescribed [1]. For comparison, we also included [¹¹¹In]Octreoscan and[¹¹¹In]DOTATOC into our study.

Internalisation and externalisation experiments were performed using thesst₂ expressing rat pancreatic tumor cell line AR42J. Free, surfacebound and internalised radioactivity were determined after 10, 30, 60,90 and 120 min. of incubation with the radioligands at 37° C. In theexternalisation experiments allowing ligand recycling, the fraction ofradioactivity released into the supernatant during 10, 30, 60, 90 and120 min as well as the radioactivity remaining in the cells weredetermined. We also measured the activity released during 5 subsequentincubations (10, 20 and 3×30 min) with intermediate changes of themedium (limited recircling).

The influence of carbohydration of TOC on the internalization parallelsthe size of the sugar used. Compared to [¹²⁵]TOC only the glucoseconjugate showed enhanced uptake in the cells. In contrast,intracellular accumulation of both [¹²⁵I]Gluc-TOCA and [¹²⁵I]Mtr-TOCA(FIG. 3 and Tab.2) was significantly increased (7.36±0.50 and 5.68±0.38fold compared to [¹²⁵I]TOC). Comparing the internalisationcharacteristics of TOC and TOCA and of the respective glucosylatedderivatives, we observed a synergistic effect of both structuralmodifications (substitution of Thr (ol)⁸ by Thr⁸ and carbohydration) onsurface availability and on internalisation rate.

Compared to [¹¹¹In]Octreoscan and [¹¹¹In]DOTATOC, the internalization ofboth ^(99m)Tc-labelled carbohydrated derivatives was unexpectedly high.

Pretreatment experiments with 10 μM sandostatin reduced the liganduptake to a max. of 5% of control, indicating i) sst₂ specific uptakeand ii) that no carbohydrate related uptake mechanism is involved(Tab.2). For all tracers investigated, the internalized activitystrongly correlates with the suface availability of the ligands. TABLE 2Internalisation data obtained after a 60 min incubation with AR42J cellsInternalisation values as well as ratios are normalized to the reference[¹²⁵I] TOC internalised surface internalisation ligand availabilityrate^(c) [¹²⁵I] Mtr-TOC  42 ± 7*  47 ± 11 87 ± 8 [¹²⁵I] Malt-TOC 66 ± 4 68 ± 11  95 ± 18 [¹²⁵I] TOC 100 100 100 [¹²⁵I] Gluc-TOC 143 ± 14 136 ±13 110 ± 12 [¹²⁵I] TOCA 204 ± 7* 182 ± 6  140 ± 8  [¹²⁵I] Gluc-TOCA  736± 50* 583 ± 53 196 ± 21 [¹²⁵I] Mtr-TOCA  568 ± 38* 312 ± 25 216 ± 26[¹¹¹I] Octreoscan 16 ± 2 24 ± 2  63 ± 12 [¹¹¹I] DOTATOC 30 ± 7 28 ± 7 55 ± 11 [^(99m)Tc] Gluc-K⁰ (H)-TOC 234 ± 24 204 ± 54 171 ± 29[^(99m)Tc] Mtr-K⁰ (H)-TOC 298 ± 37 195 ± 35 186 ± 14^(a)surface availability = % surface-bound ligand/% free radioligand^(b)internalisation rate = % internalised ligand/% surface-bound ligand*pre-treatment experiments with 10 M sandostatin reduced cellular liganduptake by a minimum of 95%With respect to externalisation glycosylation seems to have no influence(FIG. 4). The degree of externalisation of radioligand from the cells isprimarily determined by the nature of the peptide (TOC or TOCA) and alsoby the intracellular fate of the label. In the externalisationexperiment that allowed ligand recycling, TOCA and its glycatedderivatives showed about ⅔ of extracellularly located radioactivitycompared to the experiment without recycling, while about 80% were foundfor TOC, Gluc-TOC and Mtr-TOC. [^(99m)Tc]Gluc-K⁰(H)-TOC as well as both¹¹¹In-labelled compounds showed nearly quantitative retention in thecell within 120 min under recycling conditions. In contrast,intracellular activity of [¹¹¹In]Octreoscan and (¹¹¹In]DOTATOC wasdepleted by 33% and 49% during 120 min, when recycling was limited.

Example 4

[Tc-99 m] (CO)₃-Labeled Carbohydrated SSTR-Ligands: Synthesis,Internalization Kinetics and Biodistribution on a Rat Pancreatic TumorModel.

Aim: This study is to demonstrate the design of tracers for specific andhigh-level targeting of SSTR expressing tumors. For this purpose weintroduced carbohydrated octreotide derivatives to optimizepharmacokinetics and to improve SSTR specific internalization and tumorretention. Here we describe compounds of a series of new carbohydrated^(99m)Tc-SSTR ligands.

Methods: Coupling of glucose (Gluc) and maltotriose (Mtr) to N-Lys⁰ ofN-(Boc-His(Boc))Lys⁰-Tyr³-Lys⁵(Dde)-octreotide (protected K(H)TOC) andfinal deprotection yielded Gluc- and Mtr-K(H)TOC. ^(99m)Tc-labeling wascarried out using [^(99m)Tc(H₂O)₃(CO)₃]⁺. Internalization andexternalization studies (<2 h) were performed using the SSTR₂ expressingrat pancreatic cell line AR42J and [¹²⁵I]Tyr³-octreotide ([¹²⁵I]TOC) asa reference. Biodistribution was evaluated in nude mice bearing AR42Jtumors at 0.5 and 2 h p.i (n=4).

Results: Radiolabeling produced efficiently (0.5 h, RCY>97%) uniformproducts. Compared to [¹²⁵I]TOC, both carbohydrated peptides showedsignificantly higher intercellular activity levels; e.g. at 2 h 2.3±0.2times higher levels for [^(99m)Tc]Gluc-K(H)TOC and 3.6±0.4 times higherlevels for [^(99m)Tc]Mtr-K(H)TOC. Externalization studies revealed adecrease of about 50% of intracellular activity for [¹²⁵I]TOC within 2h, while the intercellular level of [^(99m)Tc]Gluc-K(H)TOC was almoststable up to 2 h, demonstating retention of this tracer. In vivo, tumoraccumulation reached 12.2±1.0 and 14.0±6.3% ID/g at 2 h p.i. with bloodlevels of 1.5±0.2% ID/g and 4.4±1.0% ID/g for [^(99m)Tc]Gluc-K(H)TOC(T/liver 0.77, T/kidney 0.63) and [^(99m)Tc]Mtr-K(H)TOC (T/liver 0.97,T/kidney 0.84), respectively.

Conclusion: These results demonstrate the usefulness of carbohydrationas a tool to improve SSTR targeting.

Example 5

[Tc-99m] (CO)₃-Labeled Carbohydrated SSTR-Ligands: Synthesis,Internalization Kinetics and Biodistribution on a Rat Pancreatic TumorModel

Aim: This study is part of our endeavor to design tracers for specificand high-level targeting of SSTR expressing tumors. For this purpose weuse suitable carbohydrate moietics to optimize the pharmacokinetics andto improve SSTR specific internalization and tumor retention. Here wedescribe for the first time carbohydrated SSTR binding peptides labeledvia the [^(99m)Tc (H₂O)₃(CO)₃]⁺ aquaion approach.

Methods: N (Boc-His(Boc))Lys⁰-Tyr³-Lys⁵ (Dde)-octreotide (protectedK(H)TOC) was synthesized using standard Fmoc-chemistry. Coupling ofglucose (Gluc) and maltotriose (Mtr) to N-Lys⁰ and final deprotectionyielded the Gluc- and Mtr-derivatives of K(H)TOC. Tc-99m-labeling wascarried out using the organometallic aquaion [^(99m)Tc(H₂O)₃(CO)₃]⁺ (1).Internalization and externalization studies (<120 min) were performedfor both compounds using the SSTR₂ expressing cell line AR42J. For thein vitro studies [125 i]Tyr³-octreotide ([125I]TOC) was used as areference. Biodistribution of intravenously administered[^(99m)Tc]Gluc-K(H)TOC and [^(99m)Tc]Mtr-K(H)TOC was evaluated in nudemice bearing AR42J rat pancreatic tumors at 30 and 120 min p.i (n=4).

Results: The labeling procedure produced efficiently (30 min, RCY>97%)uniform products (HPLC). Compared to the reference [125I]TOC, bothcarbohydrated peptides were significantly higher internalized at alltime points investigated; e.g. at 120 min 2.3±0.2 times higher levelsfor [^(99m)Tc]Gluc-K(H)TOC and 3.6±0.4 times higher levels for[^(99m)Tc]Gluc-K(H)TOC and 3.6±0.4 times higher levels for[^(99m)Tc]Mtr-K(H)TOC. Furthermore, externalization studies revealed adecrease of about 50% of intracellular activity for the referencecompound within 120 min, while the high level of intracellular[^(99m)Tc]Gluc-K(H)TOC was almost stable up to 2 h, demonstratingretention of this tracer in the cell line investigated. In vivo, tumoraccumulation reached 12.2±1.0 and 14.0±6.3% ID/g at 120 min p.i. withcorresponding blood activity levels of 1.5±0.2% ID/g and 4.4±1.0% ID/gfor [^(99m)Tc]Gluc-K(H)TOC (T/liver 0.77, T/kidney 0.63) and[^(99m)Tc]Mtr-K(H)TOC (T/liver 0.97, T/kidney 0.84), respectively.

Conclusion: These results demonstrate the usefulness of carbohydrationas a tool to improve SSTR targeting.

Example 6

Solid Phase Peptide Synthesis

1. Fmoc-Lys⁰-Tyr³-Lys(Dde)⁵-octreotide (Fmoc-Lys⁰-TOC(Dde))Fmoc-Thr(tBu)-ol

A solution of Fmoc-Thr(tBu)-OH (1.391 g, 3.5 mmol) in 30 ml oftetrahydrofurane (THF) was cooled to −10° C. N-methylmorpholine (386 ul,3.5 mmol) and ethylchloroformate (333 ul, 3,5 mmol) were addedsuccessively. The reaction mixture was stirred at −10° C. for 30 min.Then sodium borohydride (396 mg, 10.5 mmol) was added. Over a period of30 min 60 ml of methanol were slowly added to the reaction mixture,which was then stirred at 0° C. for 3 hours.

After the addition of 50-70 ml of 1 N HCl (the cloudy reaction mixturehas to become transparent) the organic solvents were evaporated and theremaining aequous phase was extracted twice with dichloromethane (DCM).Extraction efficiency was monitored by thin layer chromatography(product R_(f=)0.83 in ethyl acetae (AcOEt)) of both phases. Thecombined organic layers were dried over magnesium sulfate, filtered andconcentrated in vacuo to yield a yellowish oil. The crude product waspurified by flash chromatography using AcOEt. The product was obtainedas an oil (1.19 g, 89%).

Calculated monoisotopic mass for C₂₃H₂₉NO₄=383.21; found:m/z=406.1[M=Na]⁺

SPPS of Fmoc-Lys⁰-Dphe¹-Cys²-Tyr³-DTrp⁴-Lys (Dde)⁵-Thr⁶-Cys⁷-Thr-ol⁸

DHP-HM (3,4-Dihydro-2H-Pyran-2-ylmethoxymethylpolystyrol)-resin (1.000g, 0.94 mmol/g) was allowed to preswell in 10 ml of dry DCE(1.20Dichloroethane) for 1 hour. Then a solution of Fmoc-Thr(tBu)-ol(1.266 g, 3.3 mmol) and of Pyridinium-p-toluenesulfonate (414 mg, 1.75mmol) in 7 ml of dry DCE was added and the reaction mixture was stirredovernight at 80° C. under argon. To cap free functional groups on theresin 5 ml of phridine were added and the suspension was stirred foranother 15 min at room temperature. The loaded resin was then filteredoff, washed twice with DMF (N,N-Dimethylformamide) and with DCM anddried in an exsiccator. Final load of the resin was 0.834 mmol/g. Thepeptide sequence was assumbled on the resin bound amino alcohol usingstandard Fmoc-protocol. Briefly, the N-terminal Fmoc was removed with20% piperidine in DMF, and the resin of resin-supported peptide waswashed with NMP (N-Methylpyrrolidone). Coupling was carried out in NMPby reacting 1.5 eq of amino acid derivative with 1.5 eq ofHOBt(1-Hydroxybenzotriazol), 1.5 eq of TBTU(O-(1H-benzoltriazol-1-yl)-N,N,N′,N′-Tetramethyluronium-Tetrafluoroborate)and 3.5 eq of DIPEA (N-Ethyl-diisopropylamine). After adding theactivated amino acid to the reaction vessel, the resin was agitated foran appropriate amount of time (usually 40-60 min). The resin was thenwashed with additional NMP. The sequence of addition for the synthesisof TOC was Fmoc-Cys(Trt), Fmoc-Thr(tBu), Fmoc-Lys(Dde), Fmoc-DTrp,Fmoc-Tyr, Fmoc-Cys(Trt), Fmoc-Dphe and Fmoc-Lys(Boc). After the couplingof the last amino acid to the sequence, the resin was washed severaltimes with NMP and DCM. Dleavage from the resin and deprotection of theThr-, Cys- and Lys⁰-sidechains was performed using a mixture of TFA(trifluoroacetic acid)/TIBS (triisobutylsilane)/water (95:2.5:2.5) andDCM (1:1). After 90 min the resin was filtered and washed twice withDCM. The combined filtrates were concentrated using a rotary evaporatorand the crude product was precipitated with ether. We obtained 828 mg ofcrude produce. Yield: 83%

The crude peptide was resuspended in 50 ml of THF (per 300 mg ofpeptide), and 5 mM ammonium acetate was added, until a clear solutionwas obtained. The pH was adjusted to 7 by dropwise addition of asaturated solution of NaHCO₃. Then 100 ul (per 300 mg of peptide) ofhydrogen peroxide (30%) were added. After 30 min of stirring at roomtemperature cyclisation was complete (gradient HPLC-control: 30-80% B in15 min). The solvents were evaporated, and the cyclised product waslyophilized. Yields were quantitative.

Calculated monoisotopic mass for C₈₀H₁₀₀N₁₂O₁₆S₂=1548.68; found:m/z=1549.5 [M+H]⁺, m/z=1571.5 [M+Na]⁺

2. Fmoc-Lys⁰-Tyr³-Lys(Dde)⁵-octreotide (Fmoc-Lys⁰-TATE(Dde))Fmoc-Lys⁰-Dphe¹-Cys²-Tyr³-DTrp⁴-Lys(Dde)⁵-Thr⁶-Cys⁷-Thr⁸

To 1.502 g of dry TCP-resin a solution of 894 mg (2.25 mmol) ofFmoc-Thr(tBu) and 321 ul (1.87 mmol) of DIPEA in 15 ml of dry DCM wasadded. After stirring for 5 min, another 642 up (3.76 mmol) of DIPEAwere added. After vigorous stirring for 90 min at room temperature, 1.5ml of methanol were added to cap unreacted Tritylchloride groups. After15 min the resin was filtered, washed twice with DCM, DMF and methanolrespectively and dried in vacuo. Final load of the resin was 0.77mmol/g. Synthesis of the peptide sequenceCys(Trt)-Thr(tBu)-Lys(Dde)-DTrp-Tyr-Cys(Trt)-Dphe-Lys(Boc)-Fmoc on theresin-bound amino acid, deprotection and cleavage from the resin as wellas cyclisation were performed as described for Fmoc-Lys⁰-TOC(Dde).

Calculated monoisotopic mass for C₈₀H₉₈N₁₂O₁₇S₂=1562.66; found:m/z=([M+H]⁺)

Solution Phase Derivatization

1. Lys0(N_(ε)-Boc-His (Boc))-TOC(Dde)

A solution of 500 mg (0.32 mmol) of Fmoc-Lys⁰-TOC(Dde), 170 mg (0.48mmol) of Boc-His(Boc), 65 mg (0.048 mmol) of HOBt, 154 mg (0.48 mmol) ofTBTU and 300 ul (1.75 mmol) of DIPEA in 3 ml of DMF was stirred at roomtemperature. RP-HPLC analysis (gradient: 30-80% B in 15 min) revealedthat the reaction was complete within 30 min. The product wasprecipitated using water and dried in vacuo. To remove the N-terminalFmoc-protecting group, the peptide was dissolved in 1 ml piperidine/DMF(1:4) and stirred for 15 min. Precipitation with ether afforded thedeprotected product with a yield of 86%.

Calculated monoisotopic mass for C₈₁H₁₁₃N₁₅O₁₉S₂=1663.78; found:m/z=1873.6 [M+H]⁺, m/z=1895.7 [M+Na]⁺

2. Lys⁰-N-Amadori Derivatives of Lys⁰(Boc-His(Boc))-TOC(Dde)

Based on a method reported previously¹, 1 equiv. ofLys⁰-(Boc-His(Boc))-TOC(Dde) and 10 equiv. of the respective aldose(Glucose=S1, Maltose=S2, Maltotriose=S3) were dissolved inmethanol/acetic acid (95:5), and the reaction mixture was stirred at 60°C. for 24-48 h. The solvents were then evaporated, and the crude productwas precipitated by the addition of ether.

To remove the Dde-protection group, the glycosylated peptide wasredissolved in a small volume of DMF, 2 vol % of hydrazine hydrate wereadded and the solution was stirred at room temperature for 15 min. Thedeprotected peptide was precipitated by the addition of ether, washedeith ether and dried in vacuo. Boc-protecting groups on the His-residuewere then removed redissolving the crude products in a small volume ofTFA/TIBS/water (95:2.5:2.5) and stirring for 15 min at room temperature.The products were precipitated using either and purified via preparativeRP-HPLC (gradient: 10-60% B in 30 min). Yields ranged from 17-32%.

S1-Lys⁰(His)TOC(N_(α)-(1-deoxy-D-fructosyl)-Lys⁰N-His)-Tyr³-octreotide):

Calculated monoisotopic mass for C₆₇H₉₅N₁₅O₁₈S₂=1461.60; found:m/s=1873.6 [M+H]⁺, m/z=1895.7 [M+Na]⁺

S2Lys⁰(His)TOC(N-(O-D-glucopyranosyl-(1-4)-1-deoxy-D-fructosyl)-Lys⁰(N_(ε)-His)-Tyr3-octreotide):

Calculated monoisotopic mass for C₇₃H₁₀₅N₁₅O₂₃S₂=1623.70; found:m/z=1873.6 [M+H]⁺, m/z=1895.7 [M+Na]⁺

S3Lys⁰(His)TOC(N_(α)-(O-_(α)-D-glucopyranosyl-(1-4)-O-α-D-glucopyranousyl-(1-4)-1-deoxy-D-fructosyl)-Lys⁰(N-His)-Tyr3-octreotide:

Calculated monoisotopic-mass for C₇₉H₁₁₅N₁₅O²⁸S²=1785.75; found:m/z=1873.6 [M+H]⁺, m/z−1895.7 [M+Na]⁺

3. Lys0(N-2-picolylamino- . . . )TATE(Dde)

2-Picolylamine N,N-diacetic acid²

Imino diacetic acid (3.25 g, 24.4 mmol) was dissolved in a mixture of 30ml of absolute ethanol and a solution of 1.95 g of NaOH (49 mmol) in 10ml of water. After the addition of 4.00 g of picolylchloride (24.4 mmol)in 9 ml of water and of 1.95 g of NaOH (49 mmol) in 4 ml of water, thesolution was stirred at 70° C. for 2 h. Then another 1.95 g (49 mmol) ofNaOH were added and the solution was stirred for another 1.5 h at 70° C.TLC reaction control (AcOEt) revealed complete disappearance of thereactants at that time. The solvents were evaporated in vacuo, and theresidue was redissolved in 25 ml of water. After adjusting the pH of thesolution to 1.5 with concentrated hydrochloric acid, the solution wasagain evaporated to dryness. By several successive steps of resuspensionof the residue with methanol under reflex, hot filtration of thesuspension and concentration of the filtrate in vacuo, excess NaCl wassuccessfully removed. The picolylamino diacetic acid crstallized fromthe remaining methanolic solution overnight at −20° C.

Calculated monoisotopic mass for C₁₀H₁₂N₂O₄=224.08; found: m/z=[M=H]⁺(disodium salt)

Coupling of 2-picolylamine N,N-diacetic acid to Fmoc-Lys⁰-TATE(Dde)

A solution of 52 mg (0.19 mmol) of 2-picolylamine N,N-diacetic acid, 26mg (0.19 mmol) of HOBt, 62 mg (0.19 mmol) of TBTU and 176 μl (1.75 mmol)of DIPEA in 2 ml of DMF was stirred at room temperature for 10 min. Thissolution was added dropwise to a solution of 200 mg (0.13 mmol) ofFmoc-Lys⁰-TATE(Dde) in 1 ml of DMF under vigorous stirring. After 30 minthe product was precipitated using diethyl either and dried in vacuo. Toremove the N-terminal Fmoc-protecting group, the peptide was dissolvedin 2 ml piperidine/DMF (1:4) and stirred for 10 min. Precipitation withether afforded the deprotected product with a yield of 84%.

Calculated monoisotopic mass for C₇₅H₉₈N₁₄O₁₈S₂=1546.66; found:m/z=[M=H]⁺, m/z=[M+Na]⁺

4. Lys⁰-N_(a)-Amadori Derivatives of Lys⁰ (Pic)TATE(Dde)

Conjugation of Lys⁰(Pic)TATE(Dde) with glucose and subsequentDde-deprotection were carried out as described for theLys⁰-N_(a)-Amadori derivatives of Lys⁰(Boc-His(Boc))-TOC(Dde).

Calculated monoisotopic mass for C₇₁H₉₆N₁₄O₂₁S₂=1544.63; found:m/z=[M=H]⁺, m/z=[M+Na]₊

Radiolabelling

Based on the organometallic aquaion [^(99m)Tc(H₂O)₃(Co)₃]⁺-methodreported recently³, 10 mg of NaBH4, 8 mg of Na₂Co₃ and 41.5 mg of Na/Ktartrate were dissolved in 600 μl of [^(99m)Tc]pertechnetate eluate in aglass flask. The flask was then connected to a CO-filled balloon andsealed tightly. After stirring at 75° C. for 45 min, the solution wascooled on an ice bath and 150 μl of phosphate buffered saline (PBS,pH-7.4) were added. To destroy the reducing agent, 600 μl of 1 N HClwere added. Then the solution was neutralized with 500 μl of a saturatedsolution of NaHCO3. The presence of the [^(99m)Tc(H₂O)₃(CO)₃]⁺-aquaionwas confirmed via TLC (5% conc. HCl in methanol).

After adding 1 ml of the [^(99m)Tc(H₂O)₃(CO)₃]⁺-solution (51 MBq) to 700μg of s1-Lys⁰(His)TOC, the mixture was heated to 75° C. for 30 min.Gradient HPLC purification afforded the radiochemically pure peptideN_(a)-(1-deoxy-D-fructosyl)-Lys⁰(N-His [^(99m)Tc(H₂O)(CO)₃]+)-Tyr³-octreotide with a radiochemical yield of 34%.

S3-Lys⁰(His)TOC was labelled in an analogous manner.

Determination of Lipophilicity

To a solution of app. 300000 cpm of radiolabeled peptide in 500 ml ofPBS (pH 7.4), 500 ml of octanol were added (n=6). Vials were vortexedfor 3 min and then centrifuged at 15000 rpm for 6 min. Aliquots of 100ml of PBS and octanol were measured in a γ-Counter.

s1-Lys⁰(His)TOC: log Pow=−0.673±0.064

S3-Lys⁰(His)TOC: log Pow=−1.304±0.040

In Vitro Experiments

AR42J cells were maintained in RPMI 1640, supplemented with 10% FCS and2 mM L-glutamine.

Internalization Studies

Six-well-plates with a cell population of 8×10⁵-5×10⁶ AR42J-cells perwell were incbated at 37° C. with fresh culture medium (RPMI 1640, 1%BSA, 2 mM L-glutamine) for 1 h prior to the experiment. Then app. 300000cpm of radiometallatedpeptie in 10 μl of culture medium were added toeach well (triplicate experiments for the reference compound (intenalstandard) [¹²⁵I]TOC and for the radiometallated compound of interesteach). After incubating for 10, 30, 60, 90 or 120 min. At 37° C. thesupernatant was collected and the cells were washed once with 1 ml ofcold medium. The supernatant was pooled with the supernatant of theprevious step. To remove recpetor bound (acid releasable) radioactivitythe cells were incubated at 37° C. with 1 ml of 0.02 M NaOAc (acid wash)After 10 min. The supernatant was collected, the cells were washed oncewith 1 ml of 0.02 M NaOAc and the supernatant was again pooled with thesupernatant of the previous step. Then the cells were extracted with 1ml of 1 N NaOH and the wells were rinsed once with PBS. This pooledfraction represents internalized radioligand. The activity in theNaOH-fraction as well as in the supernatant- and in the acidwash-fractions was cuonted in a γ-Counter.

Externalization Studies

As described for the internalization studies, the six-well-plates wereincubated at 37° C. with resh culture medium (RPMI 1640, 1% BSA, 2 mML-Glutamine) for 1H prior to the experiment. Then app. 300000 cpm ofradiometallated peptide in 10 μl of culture medium were added to eachwell (triplicate experiments for the reference compound (internalstandard) [¹²⁵I]TOC and for the radiometallated compound of interesteach). After incubating for 120 min. At 37° C., the supernatant wascollected and the cells were washed once with 1 ml of cold medium. Thesupernatant was pooled with the supernatant of the previous step. Tocollect externalized radioactivity the cells were then incubated at 37°C. with 1 ml of culture medium. After 10, 30, 60, 90 and 120 min.Respectively the supernatant was collected, the cells were washed oncewith 1 ml of culture medium and the supernatant was again pooled withthe supernatant of the previous step. Then the cells were extracted with1 ml of 1 N NaOH and the wells were rinsed once with PBS. This pooledfraction respresents radioligand still located in the cell. The activityin the NaOH-fraction as well as in the supernatant- and in theexternalisation-fractions was counted in a γ-Counter.

Externalization Studies Without Recycling of the Tracer

As described for the previous experiments six-well plate was incubatedfor 120 min. At 37° C. with radioligand. The supernatant was collectedand the cells were washed once with 1 ml of cold medium. The supernatantwas pooled with the supernatant of the previous step. To collectexternalized radioactivity the cells were then incubated at 37° C. with1 ml of culture medium. After 10 min. The supernatant was collected andthe cells were again incubated with 1 ml of culture medium for another20 min. At 370C. The supernatant was collected and the cells were threemore time incubated with 1 ml of culture medium for 30 min. At 370C.Then the cells were extracted with 1 ml of 1 N NaOH and the wells wererinsed once with PBS. This pooled fraction represents radioligand stilllocated in the cell. The activity in the NaOH-fraction as well as in theexternalisation-fractions at all time points was counted in a γ-Counter.

Biodistribution Studies

To establish tumor growth, subconfluent monolayer cells were treatedwith 1 mM EDTA, suspended, centrifuged and resuspended in RPMI 1640.

Nude mice (male and female, 6-8 weeks) were inoculated s.c. in the flankwith 2.5-3×10⁷ ells suspended in 100 μl of PBS (pH 7.4), were injectedi.v. into the tail vein of nude mice bearing an AR42J tumor. The animals(n=3-4 were sacrificed 30 and 120 min p.i, and the organs of interestwere dissected. The radioactivity was measured in weighted tissuesamples using a gamma counter. Data are expressed in % iD/g tissue(mean±DS, n=3-4). TABLE 1 Tissue accumulation [% iD/g] Of [^(99m)Tc]S1-Lys⁰ (His) TOC And [^(99m)Tc] S3-Lys⁰ (His) TOC In AR42J tumorbearing Nude mice 2 h p.i. (n = 3-4) [^(99m)] Tc] S1-Lys⁰ (His) TOC[^(99m)] Tc] S3-Lys⁰ (His) TOC blood 1.49 ± 0.18 4.12 ± 0.87 liver 15.89± 2.70  14.34 ± 1.55  intestine 2.83 ± 0.28 2.02 ± 0.41 stomach 11.28 ±2.74  6.22 ± 0.75 kidney 19.49 ± 2.22  17.97 ± 2.76  muscle 0.22 ± 0.040.40 ± 0.10 adrenals 6.47 ± 1.46 4.94 ± 0.38 pancreas 6.43 ± 2.09 3.32 ±0.38 tumor 12.21 ± 0.96  14.02 ± 6.33 

1. A somatostatin receptor (sst) binding peptidic ligand comprisingnatural or unnatural amino acids or a peptidomimetic structure, at leastone carbohydrate and at least one chelating group allowing mono- ormultidentate complexation of a radioisotope selected from Tc and Re. 2.The ligand of claim 1 wherein the radioisotope is an isotope of Tc. 3.The ligand of claim 1 wherein the chelating group is bound to theN-terminal of the peptide and the carbohydrate is bound to the chelatinggroup.
 4. The ligand of claim 1 wherein the carbohydrate is bound to theN-terminal of the peptide and the chelating group is bound to thecarbohydrate.
 5. A ligand of claim 1 wherein the carbohydrate is asugar.
 6. A ligand of claim 5 wherein the sugar is a mono- orpolysaccharide.
 7. A ligand of claim 1 further comprising at least onemultifunctional linker unit.
 8. A ligand of claim 7 wherein the sugar iscoupled to the receptor binding structure, to the multifunctional linkerunit or to the chelator via a glycosidic bond
 9. A ligand of claim 7wherein the sugar is a coupling product between an amino group in thereceptor binding structure, the multifunctional linker unit or thechelator and an aldose via Amadori Reaction.
 10. A ligand of claim 7wherein the sugar selected from the group consisting of glucose, maltoseand maltotriose.
 11. A ligand of claim 7 wherein the ligand isglycosylated at the N-terminal.
 12. The ligand of claim 7 wherein theradioisotope is selected form the group consisting of isotopes Tc andRe.
 13. A ligand of claim 7 wherein the multifunctional linker unit iscombined with the N terminus of the peptide, and the carbohydrate andthe chelating group are bound to the multifunctional linker unit.
 14. Aligand of claim 7 wherein the multifunctional linker unit is bound tothe N-terminal of the ligand, the chelating group is bound to themultifunctional linker unit and the carbohydrate is bound to thechelating group.
 15. The ligand of claim 1 further comprising at leastone multifunctional linker unit and at least two sst-receptor bindingstructures peptide groups.
 16. The ligand of claim 14 wherein the twopeptide groups are bound to the multifunctional linker unit and both thechelating group and the carbohydrate are bound to the multifunctionallinker.
 17. The ligand of claim 14 wherein there are two carbohydrateunits, each bound to the N-terminal of one of the peptide units, each ofsaid carbohydrate units bound to a multifunctional linker unit and thechelating group is bound to the multifunctional linker unit.
 18. Theligand of claim 14 wherein there are two multifunctional linker units,each bound to the N-terminal of the peptide, one chelating group linkingthe multifunctional linker units and two carbohydrate groups, each boundto one of the multifunctional linker units.
 19. The ligand of claim 14wherein the radioisotope is selected from the group consisting ofisotopes of Tc and Re.
 20. A ligand of claim 14 wherein the carbohydrateis the sugar is a coupling product between an amino group in thereceptor binding structure, the multifunctional linker unit or thechelator and an aldose via Amadori Reaction.
 21. A ligand of claim 14wherein the sugar is coupled to the receptor binding structure, to themultifunctional linker unit or to the chelator via a glycosidic bond.22. A pharmaceutical composition comprising a composition of claim 1 anda pharmaceutically acceptable carrier.
 23. A pharmaceutical compositioncomprising a composition of claim 7 and a pharmaceutically acceptablecarrier.
 24. A pharmaceutical composition comprising a composition ofclaim 14 and a pharmaceutically acceptable carrier.
 25. A method ofimaging selected from the group consisting of sst-imaging whichcomprises administering to a patient a composition of claim
 22. 26. Amethod of imaging selected from the group consisting of sst-imagingwhich comprises administering to a patient a composition of claim 23.27. A method of imaging selected from the group consisting of sst-magingwhich comprises administering to a patient a composition of claim 24.28. The compound [^(99m)Tc]Gluc-K⁰(H)TOC.
 29. The compound[^(99m)Tc]Mtr-K⁰(H)TOC.